Custom antibody development

We offer world-leading antibody development platforms based on a proprietary RabMAb® rabbit monoclonal platform, a phase display platform (AxioMx), and a next-generation sequencing platform (NGS-RabMAb®)

The antibody was affinity purified from rabbit antiserum by affinity chromatography using epitope specific phosphopeptide. The antibody against non phosphopeptide was removed by chromatography using non phosphopeptide corresponding to the phosphorylation site.

Applications

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application

Abreviews

Notes

WB

1/500 - 1/1000. Predicted molecular weight: 57 kDa.

IHC-P

Use at an assay dependent concentration.

ELISA

1/20000.

ICC/IF

Use a concentration of 1 - 5 µg/ml.

Target

Function

DNA-binding protein that specifically binds heat shock promoter elements (HSE) and activates transcription. In higher eukaryotes, HSF is unable to bind to the HSE unless the cells are heat shocked.

Sequence similarities

Belongs to the HSF family.

Domain

the 9aaTAD motif is a transactivation domain present in a large number of yeast and animal transcription factors.

Post-translationalmodifications

Phosphorylated on multiple serine residues, a subset of which are involved in stress-related regulation of transcription activation. Constitutive phosphorylation represses transcriptional activity at normal temperatures. Levels increase on specific residues heat-shock and enhance HSF1 transactivation activity. Phosphorylation on Ser-307 derepresses activation on heat-stress and in combination with Ser-303 phosphorylation appears to be involved in recovery after heat-stress. Phosphorylated on Ser-230 by CAMK2, in vitro. Cadmium also enhances phosphorylation at this site. Phosphorylation on Ser-303 is a prerequisite for HSF1 sumoylation. Phosphorylation on Ser-121 inhibits transactivation and promotes HSP90 binding. Phosphorylation on Thr-142 also mediates transcriptional activity induced by heat. Phosphorylation on Ser-326 plays an important role in heat activation of HSF1 transcriptional activity.Sumoylated with SUMO1 and SUMO2 on heat-shock. Heat-inducible sumoylation occurs after 15 min of heat-shock, after which levels decrease and at 4 hours, levels return to control levels. Sumoylation has no effect on HSE binding nor on transcriptional activity. Phosphorylation on Ser-303 is a prerequisite for sumoylation.

ab47369 (1/50) staining HSF1 in paraffin-embedded human breast carcinoma tissue. Right-hand panel corresponds to a negative control for which ab47369 was pre-incubated with the immunising (blocking) peptide.

ICC/IF image of ab47369 stained HepG2 cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab47369, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.